Soybean seed and oil compositions and methods of making same

ABSTRACT

Soybean oil compositions with unique fatty acid profiles are disclosed. These oils can be derived by the suppression of endogenous soybean FAD2 and FAD3 genes and the expression of a stearoyl acyl ACP thioesterase. Soybean plants and seeds comprising these oils are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/242,745, filed on Sep. 15, 2009.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

APPENDIX

Not Applicable.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named “38-77(54823_A)SEQ LIST”, which is 39,716 bytes in size (measured in MS-Windows),created in 10 Mar. 2009 is filed herewith by electronic submission andherein incorporated by reference. This Sequence Listing consists of SEQID NOs:1-7.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to modulating fatty acid profiles ofsoybean seed through genetic engineering and the resulting soybean oilcompositions. Recombinant DNA constructs, soybean plants and seeds, andsoybean oil composition with altered fatty acid profile are provided.

2. Related Art

Plant oils are used in a variety of applications. Novel vegetable oilcompositions and improved approaches to obtain oil compositions, fromsynthetic or natural plant sources, are needed. Depending upon theintended oil use, various fatty acid compositions are desired. Plants,especially species which synthesize large amounts of oils in seeds, arean important source of oils both for edible and industrial uses. Seedoils are composed almost entirely of triacylglycerols in which fattyacids are esterified to the three hydroxyl groups of glycerol.

Soybean oil typically contains about 11-17% saturated fatty acids: 8-13%palmitate and 3-4% stearate. See generally Gunstone et al., The LipidHandbook, Chapman & Hall, London (1994). Soybean oil has been modifiedby various breeding methods to create benefits for specific markets.However, for the production of most baked goods and coatings there is aneed for high solids-containing stable fats. In the past, partiallyhydrogenated soybean oil was used for this purpose, but with theintroduction of trans fatty acid labeling, the desirability of this oilhas decreased.

Higher plants synthesize fatty acids via a common metabolic pathway—thefatty acid synthetase (FAS) pathway, which is located in the plastids.β-ketoacyl-ACP synthases are important rate-limiting enzymes in the FASof plant cells and exist in several versions. β-ketoacyl-ACP synthase Icatalyzes chain elongation to palmitoyl-ACP (C16:0), whereasβ-ketoacyl-ACP synthase II catalyzes chain elongation to stearoyl-ACP(C18:0). In soybean, the major products of FAS are 16:0-ACP and18:0-ACP. The desaturation of 18:0-ACP to form 18:1-ACP is catalyzed bya plastid-localized soluble delta-9 desaturase (also referred to as“stearoyl-ACP desaturase”). See Voelker et al., 52 Annu. Rev. PlantPhysiol. Plant Mol. Biol. 335-61 (2001).

The products of the plastidial FAS and delta-9 desaturase, 16:0-ACP,18:0-ACP, and 18:1-ACP, are hydrolyzed by specific thioesterases (FAT).Plant thioesterases can be classified into two gene families based onsequence homology and substrate preference. The first family, FATA,includes long chain acyl-ACP thioesterases having activity primarily on18:1-ACP. Enzymes of the second family, FATB, commonly utilize 16:0-ACP(palmitoyl-ACP), 18:0-ACP (stearoyl-ACP), and 18:1-ACP (oleoyl-ACP).Such thioesterases have an important role in determining chain lengthduring de novo fatty acid biosynthesis in plants, and thus these enzymesare useful in the provision of various modifications of fatty acylcompositions, particularly with respect to the relative proportions ofvarious fatty acyl groups that are present in seed storage oils.

The products of the FATA and FATB reactions, the free fatty acids, leavethe plastids and are converted to their respective acyl-CoA esters.Acyl-CoAs are substrates for the lipid-biosynthesis pathway (KennedyPathway), which is located in the endoplasmic reticulum (ER). Thispathway is responsible for membrane lipid formation as well as thebiosynthesis of triacylglycerols, which constitute the seed oil. In theER there are additional membrane-bound desaturases, which can furtherdesaturate 18:1 to polyunsaturated fatty acids. A delta-12 desaturase(FAD2) catalyzes the insertion of a double bond into oleic acid (OA)(18:1), forming linoleic acid (LA) (18:2). A delta-15 desaturase (FAD3)catalyzes the insertion of a double bond into 18:2, forming alphalinolenic acid (ALA) (18:3).

Inhibition of the endogenous FAD2 genes through use of transgenes thatsilence the expression of FAD2 has been shown to confer a desirableoleic acid (18:1) phenotype (i.e. soybean seed comprising about 50% and75% oleic acid by weight). Transgenes and transgenic plants that providefor inhibition of the endogenous FAD2 gene expression and a desirableoleic phenotype are disclosed in U.S. Pat. No. 7,067,722. In contrast,soybean cultivars that lack FAD2-inhibiting transgenes typically produceseed with oleic acid compositions of less than 20%.

Soybean oil typically contains about 8% ALA (18:3) that renders this oiloxidatively unstable. The levels of ALA in soybean oil can be reduced byhydrogenation to improve both stability and flavor. Unfortunately,hydrogenation results in the production of trans-fatty acids, whichincreases the risk for coronary heart disease when consumed.

Oleic acid has one double bond, but is still relatively stable at hightemperatures, and oils with high levels of OA are suitable for cookingand other processes where heating is required. Recently, increasedconsumption of high OA oils has been recommended, because OA appears tolower blood levels of low density lipoproteins (“LDLs”) withoutaffecting levels of high density lipoproteins (“HDLs”). However, somelimitation of OA levels is desirable, because when OA is degraded athigh temperatures, it creates negative flavor compounds and diminishesthe positive flavors created by the oxidation of LA. Neff et al., JAOCS,77:1303-1313 (2000); Warner et al., J. Agric. Food Chem. 49:899-905(2001). It is thus preferable to use oils with OA levels that are 65-85%or less by weight, in order to limit off-flavors in food applicationssuch as frying oil and fried food. Other preferred oils have OA levelsthat are greater than 55% by weight in order to improve oxidativestability.

SUMMARY OF THE INVENTION

It is in view of the above problems that the present invention wasdeveloped. The invention first relates to a non-hydrogenated soybean oilcomposition comprising an oleic acid content greater than 35%, a stearicacid content greater than 10% and a linolenic acid content of less thanabout 2% of total seed fatty acids by weight. In other embodiments, thestearic acid content is greater than 17%, 25% and/or 30% of total fattyacids by weight. The invention further comprises a soybean plant capableof producing seed that yields the soybean oil composition above and aseed of the soybean plant comprising the soybean oil described above. Inother embodiments, the soybean plant with the above oil composition istransgenic. In certain embodiments, the soybean plant with the above oilcomposition comprises in its genome a DNA construct comprising a DNAsegment expressing a thioesterase with activity on stearoyl acyl ACP. Ina further embodiment, the thioesterase is encoded by a FATA gene. In yetanother embodiment, the thioesterase is a mangosteen thioesterase. In afurther embodiment, the thioesterase gene is a polynucleotide encodingthe polypeptide sequence of SEQ ID NO: 1. In certain embodiments, theDNA construct further comprises a first DNA segment expressing athioesterase with activity on stearoyl acyl ACP and a second DNA segmentdesigned to trigger the suppression of an endogenous FAD2 gene or theconstruct further comprises a first DNA segment expressing athioesterase with activity on stearoyl acyl ACP and a second DNA segmentdesigned to trigger the suppression of an endogenous FAD3 gene or theconstruct further comprises a first DNA segment expressing athioesterase with activity on stearoyl acyl ACP and a second DNA segmentdesigned to trigger the suppression of endogenous FAD2 and FAD3 genes.

The invention further relates to a soybean oil composition produced froma transgenic soybean seed wherein the soybean oil composition comprisesa stearic acid content greater than 10% and a linolenic acid content ofless than about 5% of total seed fatty acids by weight. In otherembodiments, the stearic acid content is greater than 17%, 25% and/or30% of total fatty acids by weight. The invention further comprises atransgenic soybean plant capable of producing seed that yield thesoybean oil composition immediately above and transgenic seed of thetransgenic soybean plant comprising the transgenic soybean oil describedimmediately above. In certain embodiments, the transgenic soybean plantwith the immediately above oil composition comprises a DNA constructcomprising a DNA segment expressing a thioesterase with activity onstearoyl acyl ACP. In a further embodiment, the thioesterase is encodedby a FATA gene. In yet another embodiment, the thioesterase is amangosteen thioesterase. In a further embodiment, the thioesterase geneis a polynucleotide encoding the polypeptide sequence of SEQ ID NO: 1.In certain embodiments, the DNA construct further comprises a first DNAsegment expressing a thioesterase with activity on stearoyl acyl ACP anda second DNA segment designed to trigger the suppression of anendogenous FAD2 gene or the construct further comprises a first DNAsegment expressing a thioesterase with activity on stearoyl acyl ACP anda second DNA segment designed to trigger the suppression of anendogenous FAD3 gene or the construct further comprises a first DNAsegment expressing a thioesterase with activity on stearoyl acyl ACP anda second DNA segment designed to trigger the suppression of endogenousFAD2 and FAD3 genes. The invention further relates to a soybean oilcomposition produced from a transgenic soybean seed wherein the soybeanoil composition comprises a stearic acid content greater than 20% and alinolenic acid content of less than about 1.7% of total seed fatty acidsby weight. The invention further relates to a soybean oil compositionproduced from a transgenic soybean seed wherein the soybean oilcomposition comprises a stearic acid content greater than 20% and alinolenic acid content of less than about 2% of total seed fatty acidsby weight. The invention further relates to a soybean oil compositionproduced from a transgenic soybean seed wherein the soybean oilcomposition comprises a stearic acid content greater than 32% and alinolenic acid content of less than about 5% of total seed fatty acidsby weight. The invention further relates to a soybean oil compositionproduced from a transgenic soybean seed wherein the soybean oilcomposition comprises a stearic acid content greater than 10% and anoleic acid content of about 39 to 57% of total seed fatty acids byweight. The invention further relates to a soybean oil compositionproduced from a transgenic soybean seed wherein the soybean oilcomposition comprises a stearic acid content of 10 to 28%, an oleic acidcontent of 25-57% and a linolenic acid content of less than about 3% oftotal seed fatty acids by weight.

This invention also encompasses a method for producing a soybean oilseedcrop capable of yielding the soybean oil composition comprising an oleicacid content greater than 35%, a stearic acid content greater than 10%and a linolenic acid content of less than about 2% of total seed fattyacids by weight upon heptane extraction, comprising growing a soybeanplant of the invention to maturity under plant growth conditions andharvesting seeds from said plant to form a soybean seed crop. Theinvention also encompasses a method for producing a soybean oilseed cropcapable of yielding the soybean oil composition comprising a stearicacid content greater than 10% and a linolenic acid content of less thanabout 5% of total seed fatty acids by upon heptane extraction,comprising growing a soybean plant of the invention to maturity underplant growth conditions and harvesting seeds from said plant to form asoybean seed crop.

Further features and advantages of the present invention, as well as thestructure and operation of various embodiments of the present invention,are described in detail below with reference to the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthe specification, illustrate the embodiments of the present inventionand together with the description, serve to explain the principles ofthe invention. In the drawings:

FIG. 1 illustrates the plant vector pMON97616;

FIG. 2 illustrates the plant vector pMON97617;

FIG. 3 illustrates the plant vector pMON97620;

FIG. 4 illustrates the plant vector pMON97623; and

FIG. 5 illustrates the plant vector pMON97624.

DETAILED DESCRIPTION OF THE INVENTION

Description of the nucleic acid sequences.

SEQ ID NO: 1 is a peptide sequence of a Garcinia mangostana FATA.

SEQ ID NO: 2 is a nucleic acid sequence of a Garcinia mangostana FATA.

SEQ ID NO: 3 is a nucleic acid sequence of the first T-DNA of pMON97616.

SEQ ID NO: 4 is a nucleic acid sequence of the first T-DNA of pMON97617.

SEQ ID NO: 5 is a nucleic acid sequence of the first T-DNA of pMON97620.

SEQ ID NO: 6 is a nucleic acid sequence of the first T-DNA of.pMON97623.

SEQ ID NO: 7 is a nucleic acid sequence of the first T-DNA of pMON97624.

DEFINITIONS

“ACP” refers to an acyl carrier protein moiety.

“Altered seed oil composition” refers to a seed oil composition from asoybean plant of the invention which has altered or modified levels ofthe fatty acids therein, relative to a typical soybean seed oil.

“Antisense suppression” refers to gene-specific silencing that isinduced by the introduction of an antisense RNA molecule.

“Agronomically elite”, as used herein, means a genotype that has aculmination of many distinguishable traits such as emergence, vigor,vegetative vigor, disease resistance, seed set, standability andthreshability which allows a producer to harvest a product of commercialsignificance.

“Allele” as used herein, refers to any of one or more alternative formsof a gene locus, all of which alleles relate to a trait orcharacteristic. In a diploid cell or organism, the two alleles of agiven gene occupy corresponding loci on a pair of homologouschromosomes.

“Backcrossing” as used herein, refers to a process in which a breederrepeatedly crosses hybrid progeny, for example a first generation hybrid(F1), back to one of the parents of the hybrid progeny. Backcrossing canbe used to introduce one or more single locus conversions from onegenetic background into another.

“Coexpression of more than one agent such as an mRNA or protein” refersto the simultaneous expression of an agent in overlapping time framesand in the same cell or tissue as another agent. “Coordinated expressionof more than one agent” refers to the coexpression of more than oneagent when the production of transcripts and proteins from such agentsis carried out utilizing a shared or identical promoter.

“Complement” of a nucleic acid sequence refers to the complement of thesequence along its complete length.

“Cosuppression” is the reduction in expression levels, usually at thelevel of RNA, of a particular endogenous gene or gene family by theexpression of a homologous sense construct that is capable oftranscribing mRNA of the same strandedness as the transcript of theendogenous gene. Napoli et al., Plant Cell 2:279-289 (1990); van derKrol et al., Plant Cell 2:291-299 (1990).

A “CP4 EPSPS” or “CP4 5-enolpyruvylshikimate-3-phosphate synthase” geneencodes an enzyme (CP4 EPSPS) capable of conferring a substantial degreeof glyphosate resistance upon the plant cell and plants generatedtherefrom. The CP4 EPSPS sequence may be a CP4 EPSPS sequence derivedfrom Agrobacterium tumefaciens sp. CP4 or a variant or synthetic formthereof, as described in U.S. Pat. No. 5,633,435. Representative CP4EPSPS sequences include, without limitation, those set forth in U.S.Pat. Nos. 5,627,061 and 5,633,435.

“Crossing”, as used herein, refers to the mating of two parent plants.

“Cross-pollination”, as used herein, refers to fertilization by theunion of two gametes from different plants.

“F₁” or “F1”, as used herein, refers to first generation progeny of thecross of two plants.

“F₁ Hybrid” or F1 Hybrid”, as used herein, refers to first generationprogeny of the cross of two non-isogenic plants.

“F₂” or “F2”, as used herein, refers to second generation progeny of thecross of two plants.

“F₃” or “F3”, as used herein, refers to second generation progeny of thecross of two plants.

“Crude soybean oil” refers to soybean oil that has been extracted fromsoybean seeds, but has not been refined, processed, or blended, althoughit may be degummed.

“CTP” refers to a chloroplastic transit peptide, encoded by the“chloroplastic transit peptide coding sequence”.

“DNA construct” refers to the heterologous genetic elements operablylinked to each other making up a recombinant DNA molecule or segment andmay comprise elements that provide expression of a DNA polynucleotidemolecule in a host cell and elements that provide maintenance of theconstruct. A plant expression cassette comprises the operable linkage ofgenetic elements that when transferred into a plant cell providesexpression of a desirable gene product or expression of moleculedesigned to trigger the suppression of an endogenous gene.

When referring to proteins and nucleic acids herein, “derived” refers toeither directly (for example, by looking at the sequence of a knownprotein or nucleic acid and preparing a protein or nucleic acid having asequence similar, at least in part, to the sequence of the known proteinor nucleic acid) or indirectly (for example, by obtaining a protein ornucleic acid from an organism which is related to a known protein ornucleic acid) obtaining a protein or nucleic acid from a known proteinor nucleic acid. Other methods of “deriving” a protein or nucleic acidfrom a known protein or nucleic acid are known to one of skill in theart.

Double-stranded RNA (“dsRNA”) and RNA interference (“RNAi”) refer togene-specific silencing that is induced by the introduction of aconstruct capable of transcribing an at least partially double-strandedRNA molecule. A “dsRNA molecule” and an “RNAi molecule” both refer to aregion of an RNA molecule containing segments with complementarynucleotide sequences and therefore can hybridize with each other andform double-stranded RNA. Such double-stranded RNA molecules arecapable, when introduced into a cell or organism, of at least partiallyreducing the level of an mRNA species present in a cell or a cell of anorganism. In addition, the dsRNA can be created after assembly in vivoof appropriate DNA fragments through illegitimate recombination andsite-specific recombination as described in International ApplicationNo. PCT/US2005/004681, filed on Feb. 11, 2005, which is herebyincorporated by reference in its entirety.

“Exon” refers to the normal sense of the term as meaning a segment ofnucleic acid molecules, usually DNA, that encodes part of or all of anexpressed protein.

“FAD2” refers to, depending on the context, a gene (FAD2) or encodedprotein (FAD2) capable of catalyzing the insertion of a double bond intoa fatty acyl moiety at the twelfth position counted from the carboxylterminus. Italicized capital letters refer to genes and non-italicizedcapital letters refer to proteins. FAD2 proteins are also referred to as“Δ12 desaturase” or “omega-6 desaturase”. The term “FAD2-1” is used torefer to, depending on the context, a FAD2 gene or protein that isnaturally expressed in a specific manner in seed tissue, and the term“FAD2-2” is used to refer to, depending on the context, a FAD2 gene orprotein that is (a) a different gene from a FAD2-1 gene or protein and(b) is naturally expressed in multiple tissues, including the seed.

A “FAD3”, “Δ15 desaturase” or “omega-3 desaturase” gene encodes anenzyme (FAD3) capable of catalyzing the insertion of a double bond intoa fatty acyl moiety at the fifteenth position counted from the carboxylterminus. The terms “FAD3-1, FAD3-A, FAD3-B and FAD3-C” are used torefer to FAD3 gene family members that are naturally expressed inmultiple tissues, including the seed.

A “FATA” or “long chain acyl-ACP thioesterase” refers to a gene thatencodes an enzyme (FATA) capable of catalyzing the hydrolytic cleavageof the carbon-sulfur thioester bond in the panthothene prosthetic groupof acyyl-ACP as its preferred reaction with activity primarily on18:1-ACP”.

“Fatty acid” refers to free fatty acids and/or fatty acyl groups.

“Gene” refers to a nucleic acid sequence that encompasses a 5′ promoterregion associated with the expression of the gene product, any intronand exon regions and 3′ or 5′ untranslated regions associated with theexpression of the gene product.

“Gene silencing” refers to the suppression of gene expression ordown-regulation of gene expression.

A “gene family” is two or more genes in an organism which encodeproteins that exhibit similar functional attributes, and a “gene familymember” is any gene of the gene family found within the genetic materialof the plant, e.g., a “FAD2 gene family member” is any FAD2 gene foundwithin the genetic material of the plant. An example of two members of agene family are FAD2-1 and FAD2-2. A gene family can be additionallyclassified by the similarity of the nucleic acid sequences. A gene,FAD2, for example, includes alleles at that locus. Preferably, a genefamily member exhibits at least 60%, more preferably at least 70%, morepreferably at least 80% nucleic acid sequence identity in the codingsequence portion of the gene.

“Genotype”, as used herein, refers to the genetic constitution of a cellor organism.

As used herein, “Heterologous” means not naturally occurring together.

A nucleic acid molecule is said to be “introduced” if it is insertedinto a cell or organism as a result of human manipulation, no matter howindirect. Examples of introduced nucleic acid molecules include, but arenot limited to, nucleic acids that have been introduced into cells viatransformation, transfection, injection, and projection, and those thathave been introduced into an organism via methods including, but notlimited to, conjugation, endocytosis, and phagocytosis.

“Intron” refers to the normal sense of the term as meaning a segment ofnucleic acid molecules, usually DNA, that does not encode part of or allof an expressed protein, and which, in endogenous conditions, istranscribed into RNA molecules, but which is spliced out of theendogenous RNA before the RNA is translated into a protein. An “introndsRNA molecule” and an “intron RNAi molecule” both refer to adouble-stranded RNA molecule capable, when introduced into a cell ororganism, of at least partially reducing the level of an mRNA speciespresent in a cell or a cell of an organism where the double-stranded RNAmolecule exhibits sufficient identity to an intron of a gene present inthe cell or organism to reduce the level of an mRNA containing thatintron sequence.

A “low saturate” soybean seed oil composition contains between 3.6 and 8percent saturated fatty acids by weight of the total fatty acids.

A “low linolenic” oil composition contains less than about 3% linolenicacid by weight of the total fatty acids.

A “mid-oleic soybean seed” is a seed having between 55% and 85% oleicacid by weight of the total fatty acids.

The term “non-coding” refers to sequences of nucleic acid molecules thatdo not encode part or all of an expressed protein. Non-coding sequencesinclude but are not limited to introns, promoter regions, 3′untranslated regions (3′UTRs), and 5′ untranslated regions (5′UTRs).

The term “oil composition” refers to a soybean oil with specified levelsof fatty acids.

“Phenotype”, as used herein, refers to the detectable characteristics ofa cell or organism, which characteristics are the manifestation of geneexpression.

A promoter that is “operably linked” to one or more nucleic acidsequences is capable of driving expression of one or more nucleic acidsequences, including multiple coding or non-coding nucleic acidsequences arranged in a polycistronic configuration.

“Physically linked” nucleic acid sequences are nucleic acid sequencesthat are found on a single nucleic acid molecule. A “plant” includesreference to whole plants, plant organs (e.g., leaves, stems, roots,etc.), seeds, and plant cells and progeny of the same. The term “plantcell” includes, without limitation, seed suspension cultures, embryos,meristematic regions, callus tissue, leaves, roots, shoots,gametophytes, sporophytes, pollen, and microspores. “Plant promoters,”include, without limitation, plant viral promoters, promoters derivedfrom plants, and synthetic promoters capable of functioning in a plantcell to promote the expression of an mRNA.

A “polycistronic gene” or “polycistronic mRNA” is any gene or mRNA thatcontains transcribed nucleic acid sequences which correspond to nucleicacid sequences of more than one gene targeted for suppression. It isunderstood that such polycistronic genes or mRNAs may contain sequencesthat correspond to introns, 5′UTRs, 3′UTRs, transit peptide encodingsequences, exons, or combinations thereof, and that a recombinantpolycistronic gene or mRNA might, for example without limitation,contain sequences that correspond to one or more UTRs from one gene andone or more introns from a second gene.

As used herein, the term “R₀”, “R0”, “R₀ generation” or “R0 generation”refers to a transformed plant obtained by regeneration of a transformedplant cell.

As used herein, the term “R₁” “R1”, “R₁ generation” or “R1 generation”refers to seeds obtained from a selfed transgenic R₀ plant. R₁ plantsare grown from the R₁ seeds.

A “seed-specific promoter” refers to a promoter that is activepreferentially or exclusively in a seed. “Preferential activity” refersto promoter activity that is substantially greater in the seed than inother tissues, organs or organelles of the plant. “Seed-specific”includes without limitation activity in the aleurone layer, endosperm,and/or embryo of the seed.

“Sense intron suppression” refers to gene silencing that is induced bythe introduction of a sense intron or fragment thereof. Sense intronsuppression is described, for example by Fillatti in PCT WO 01/14538 A2.

“Simultaneous expression” of more than one agent such as an mRNA orprotein refers to the expression of an agent at the same time as anotheragent. Such expression may only overlap in part and may also occur indifferent tissue or at different levels.

“Total oil level” refers to the total aggregate amount of fatty acidwithout regard to the type of fatty acid. As used herein, total oillevel does not include the glycerol backbone.

“Transgene” refers to a nucleic acid sequence associated with theexpression of a gene introduced into an organism. A transgene includes,but is not limited to, a gene endogenous or a gene not naturallyoccurring in the organism.

A “transgenic plant” is any plant that stably incorporates a transgenein a manner that facilitates transmission of that transgene from a plantby any sexual or asexual method.

A “zero saturate” soybean seed oil composition contains less than 3.6percent saturated fatty acids by weight.

A “loss-of-function mutation” is a mutation in the coding sequence of agene, which causes the function of the gene product, usually a protein,to be either reduced or completely absent. A loss-of-function mutationcan, for instance, be caused by the truncation of the gene productbecause of a frameshift or nonsense mutation. A phenotype associatedwith an allele with a loss of function mutation can be either recessiveor dominant.

A cell or organism can have a family of more than one gene encoding aparticular enzyme, and the capital letter that follows the geneterminology (A, B, C) is used to designate the family member, i.e.,FAD2-1A is a different gene family member from FAD2-1B. Similarly,FAD3-1A, FAD3-1B, and FAD3-1C represent distinct members of the FAD3-1gene family. Loss of function alleles of various genes are representedin lowercase followed by a minus sign (i.e. fad3-1b- and fad3-1c-represent loss of function alleles of the FAD3-1B and FAD3-1C genes,respectively).

As used herein, any range set forth is inclusive of the end points ofthe range unless otherwise stated.

A. Transgenes that decrease the expression of the endogenous soybeanFAD2-1 gene

Various transgenes that decrease the expression of the endogenoussoybean FAD2-1 gene can be used to practice the methods of theinvention. By suppressing, at least partially reducing, reducing,substantially reducing, or effectively eliminating the expression of theendogenous FAD2 gene, the amount of FAD2 protein available in a plantcell is decreased, i.e. the steady-state levels of the FAD2 protein arereduced. Thus, a decrease in expression of FAD2 protein in the soybeancell can result in an increased proportion of mono-unsaturated fattyacids such as oleate (C18:1). Soybean plants that contain transgenesthat decrease the expression of the endogenous soybean FAD2-1 andproduce seed with increased oleic acid are described in U.S. Pat. No.7,067,722.

Various transgenes that decrease the expression endogenous soybean FAD3gene can be used to practice the methods of the invention for productionof soybean plants with a low alpha linolenic acid phenotype. Bysuppressing, at least partially reducing, reducing, substantiallyreducing, or effectively eliminating the expression of the endogenousFAD3 gene, the amount of FAD3 protein available in a plant cell isdecreased, i.e. the steady-state levels of the FAD3 protein are reduced.Thus, a decrease of FAD3 can result in an decreased proportion ofunsaturated fatty acids such as alpha linolenic acid (18:3).

Various methods for decreasing expression of either: 1) the endogenoussoybean FAD3 gene(s) or 2) both the endogenous soybean FAD2-1 and FAD3gene(s) in soybean plants and seed are contemplated by this invention,including, but not limited to, antisense suppression, co-suppression,ribozymes, combinations of sense and antisense (double-stranded RNAi),promoter silencing, and use of DNA binding proteins such as zinc fingerproteins. The general; practice of these methods with respect to variousendogenous plant genes is described in WO 98/53083, WO 01/14538, andU.S. Pat. No. 5,759,829. Suppression of gene expression in plants, alsoknown as gene silencing, occurs at both the transcriptional level andpost-transcriptional level. Certain of these gene silencing mechanismsare associated with nucleic acid homology at the DNA or RNA level. Suchhomology refers to similarity in DNA or protein sequences within thesame species or among different species. Gene silencing occurs if theDNA sequence introduced to a host cell is sufficiently homologous to anendogenous gene that transcription of the introduced DNA sequence willinduce transcriptional or post transcriptional gene silencing of theendogenous gene. To practice this invention, DNA sequences with about70% identity over the entire length of a DNA sequence of a soybeanFAD2-1 or FAD3 coding region or non-coding region, or to a nucleic acidsequence that is complementary to a soybean FAD2-1 or FAD3 coding ornon-coding region, have sufficient homology for suppression of steadystate expression levels of FAD2-1 or FAD3 when introduced into soybeanplants as transgenes. The transgenes of the invention more preferablycomprise DNA sequences that are, over their entire length, at least 80%identical; at least 85% identical; at least 90% identical; at least 95%identical; at least 97% identical; at least 98% identical; at least 99%identical; or 100% identical to a soybean FAD2-1 or FAD3 gene codingregion or non-coding region, or to a nucleic acid sequence that iscomplementary to a soybean FAD2-1 or FAD3 gene coding or non-codingregion. The DNA sequences with the above indicated levels of identity tothe soybean FAD2-1 or FAD3 gene(s) may be coding sequences, intronsequences, 3′UTR sequences, 5′UTR sequences, promoter sequences, othernon-coding sequences, or any combination of the foregoing. The intronmay be located between exons, or located within a 5′ or 3′ UTR of aplant gene. The coding sequence is preferably a fraction of a proteinencoding frame that does not encode a protein with FAD2 or FAD3enzymatic activity. However, it is recognized that in certain instances,such as in cosuppression, DNA sequences that encode an enzymaticallyactive FAD2 or FAD3 protein can be used to decrease expression of theendogenous soybean FAD2-1 or FAD3 gene(s).

It is also understood that DNA sequences with the above indicated levelsof identity to the soybean FAD2-1 gene that are useful in the methods ofthis invention can be derived from any soybean FAD2 gene, the soybeanFAD2-1A gene, the soybean FAD2-1A intron, soybean FAD2-1B intron, thesoybean FAD2-2 gene, alleles of the soybean FAD2-1 gene, alleles of thesoybean FAD2-2 gene, and from FAD2 genes derived from other leguminousplants such as Medicago sp., Pisum sp., Vicia sp., Phaseolus sp., andPisum sp. DNA sequence with the indicated levels of identity to thesoybean FAD2-1 sequence can be derived from multiple sources. DNAsequences with the indicated levels of sequence identity can also beobtained synthetically.

In the methods of this invention, transgenes specifically designed toproduce double-stranded RNA (dsRNA) molecules with homology to theFAD2-1 gene can also induce FAD2-1 sequence-specific silencing and beused to decrease expression of the endogenous soybean FAD2-1 gene. Thesense strand sequences of the dsRNA can be separated from the antisensesequences by a spacer sequence, preferably one that promotes theformation of a dsRNA molecule. Examples of such spacer sequences includethose set forth in Wesley et al., Plant J., 27(6):581-90 (2001), andHamilton et al., Plant J., 15:737-746 (1988). In a preferred aspect, thespacer sequence is capable of forming a hairpin structure as illustratedin Wesley et al., supra. Particularly preferred spacer sequences in thiscontext are plant introns or parts thereof. A particularly preferredplant intron is a spliceable intron. Spliceable introns include, but arenot limited to, an intron selected from the group consisting of PDKintron, FAD3-1A or FAD3-1B intron #5, FAD3 intron #1, FAD3 intron #3A,FAD3 intron #3B, FAD3 intron #3C, FAD3 intron #4, FAD3 intron #5, FAD2intron #1, and FAD2-2 intron. The sense-oriented, non-coding moleculesmay be, optionally separated from the corresponding antisense-orientedmolecules by a spacer segment of DNA. The spacer segment can be a genefragment or artificial DNA. The spacer segment can be short tofacilitate forming hairpin dsRNA or long to facilitate dsRNA without ahairpin structure. The spacer can be provided by extending the length ofone of the sense or antisense molecules as disclosed in US 2005/0176670A1. Alternatively, a right-border-right-border (“RB-RB”) sequence can becreated after insertion into the plant genome as disclosed in U.S.Patent Application 2005/0183170.

The transgenes of the invention will typically include a promoterfunctional in a plant cell, or a plant promoter, that is operably linkedto an aforementioned DNA sequence that decreases expression of anendogenous soybean FAD2-1 or FAD3 gene. Design of such a vector isgenerally within the skill of the art (See, e.g., Plant MolecularBiology: A Laboratory Manual, Clark (ed.), Springer, N.Y. (1997)).However, it is recognized that constructs or vectors may also contain apromoterless gene that may utilize an endogenous promoter uponinsertion. A number of promoters that are active in plant cells havebeen described in the literature such as the CaMV 35S and FMV promoters.Enhanced or duplicated versions of the CaMV 35S and FMV 35S promoterscan also be used to express an aforementioned DNA sequence thatdecreases expression of an endogenous FAD2-1 gene (Odell et al., Nature313: 810-812 (1985); U.S. Pat. No. 5,378,619). Additional promoters thatmay be utilized are described, for example, in U.S. Pat. Nos. 5,378,619;5,391,725; 5,428,147; 5,447,858; 5,608,144; 5,608,144; 5,614,399;5,633,441; 5,633,435; and 4,633,436. In addition, a tissue specificenhancer can be used with a basal plant promoter. Basal promoterstypically comprise a “TATA” box and an mRNA cap site but lack enhancerelements required for high levels of expression.

Particularly preferred promoters for use in the transgenes of theinstant invention are promoters that express a DNA sequence thatdecreases expression of an endogenous soybean FAD2-1 or FAD3 gene orthat expresses a FATA gene in seeds or fruits. Indeed, in a preferredembodiment, the promoter used is a seed-specific promoter. Examples ofsuch seed-specific promoters include the 5′ regulatory regions from suchgenes as napin (Kridl et al., Seed Sci. Res. 1:209-219 (1991)),phaseolin, stearoyl-ACP desaturase, 7Sα, 7Sα′ (Chen et al., Proc. Natl.Acad. Sci., 83:8560-8564 (1986)), USP, arcelin, oleate 12-hydroxylasefrom Lesquerella fendleri (Broun et al., Plant J. 13: 201-210 (1998))and oleosin. Preferred promoters for expression in the seed are 7Sα,7Sα′, napin, and FAD2-1A promoters.

Constructs or vectors will also typically include a 3′ transcriptionalterminator or 3′ polyadenylation signal that is operably linked to anaforementioned DNA sequence that decreases expression of an endogenoussoybean FAD2-1 or FAD'gene or that expresses a FATA gene. Thetranscriptional termination signal can be any transcriptionaltermination signal functional in a plant, or any plant transcriptionaltermination signal. Preferred transcriptional termination signalsinclude, but are not limited to, a pea Rubisco E9 3′ sequence, aBrassica napin 3′ sequence, a tml 3′ sequence, and an Agrobacteriumtumor-inducing (Ti) plasmid nopaline synthase (NOS) gene 3′ sequence. Itis understood that this group of exemplary polyadenylation regions isnon-limiting and that one skilled in the art could employ otherpolyadenylation regions that are not explicitly cited here in thepractice of this invention.

Finally, it is also recognized that transgenes of the invention can beinserted in plant transformation vectors that also comprise genes thatencode selectable or scoreable markers. The selectable marker gene canbe a gene encoding a neomycin phosphotransferase protein, aphosphinothricin acetyltransferase protein, a glyphosate resistant5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) protein, ahygromycin phosphotransferase protein, a dihydropteroate synthaseprotein, a sulfonylurea insensitive acetolactate synthase protein, anatrazine insensitive Q protein, a nitrilase protein capable of degradingbromoxynil, a dehalogenase protein capable of degrading dalapon, a2,4-dichlorophenoxyacetate monoxygenase protein, a methotrexateinsensitive dihydrofolate reductase protein, and an aminoethylcysteineinsensitive octopine synthase protein. The corresponding selectiveagents used in conjunction with each gene can be: neomycin (for neomycinphosphotransferase protein selection), phosphinotricin (forphosphinothricin acetyltransferase protein selection), glyphosate (forglyphosate resistant 5-enol-pyruvylshikimate-3-phosphate synthase(EPSPS) protein selection), hygromycin (for hygromycinphosphotransferase protein selection), sulfadiazine (for adihydropteroate synthase protein selection), chlorsulfuron (for asulfonylurea insensitive acetolactate synthase protein selection),atrazine (for an atrazine insensitive Q protein selection), bromoxinyl(for a nitrilase protein selection), dalapon (for a dehalogenase proteinselection), 2,4-dichlorophenoxyacetic acid (for a2,4-dichlorophenoxyacetate monoxygenase protein selection), methotrexate(for a methotrexate insensitive dihydrofolate reductase proteinselection), or aminoethylcysteine (for an aminoethylcysteine insensitiveoctopine synthase protein selection). A preferred selectable marker geneis a CP4 EPSPS gene that confers resistance to the herbicide glyphosate.The scoreable marker gene can be a gene encoding a beta-glucuronidaseprotein, a green fluorescent protein, a yellow fluorescent protein, abeta-galactosidase protein, a luciferase protein derived from a lucgene, a luciferase protein derived from a lux gene, a sialidase protein,streptomycin phosphotransferase protein, a nopaline synthase protein, anoctopine synthase protein or a chloramphenicol acetyl transferaseprotein.

The above-described nucleic acid molecules are embodiments which achievethe objects, features and advantages of the present invention. It is notintended that the present invention be limited to the illustratedembodiments. The arrangement of the sequences in the first and secondsets of DNA sequences within the nucleic acid molecule is not limited tothe illustrated and described arrangements, and may be altered in anymanner suitable for achieving the objects, features and advantages ofthe present invention as described herein and illustrated in theaccompanying drawings.

Transgenic Organisms, and Methods for Producing Same

Any of the nucleic acid molecules and constructs of the invention may beintroduced into a soybean plant or plant cell in a permanent ortransient manner. Methods and technology for introduction of DNA intosoybean plant cells are well known to those of skill in the art, andvirtually any method by which nucleic acid molecules may be introducedinto a cell is suitable for use in the present invention. Non-limitingexamples of suitable methods include: chemical methods; physical methodssuch as microinjection, electroporation, the gene gun, microprojectilebombardment, and vacuum infiltration; viral vectors; andreceptor-mediated mechanisms. Other methods of cell transformation canalso be used and include but are not limited to introduction of DNA intoplants by direct DNA transfer into pollen, by direct injection of DNAinto reproductive organs of a plant, or by direct injection of DNA intothe cells of immature embryos followed by the rehydration of desiccatedembryos.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells. See, e.g., Fraley et al.,Bio/Technology 3:629-635 (1985); Rogers et al., Methods Enzymol.153:253-277 (1987). The region of DNA to be transferred is defined bythe border sequences and intervening DNA is usually inserted into theplant genome. Spielmann et al., Mol. Gen. Genet. 205:34 (1986). ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations.Klee et al., In: Plant DNA Infectious Agents, Hohn and Schell (eds.),Springer-Verlag, New York, pp. 179-203 (1985). Agrobacterium-mediatedtransformation of soybean is specifically described in U.S. Pat. No.7,002,058.

Transgenic plants are typically obtained by linking the gene of interestto a selectable marker gene, introducing the linked transgenes into aplant cell, a plant tissue or a plant by any one of the methodsdescribed above, and regenerating or otherwise recovering the transgenicplant under conditions requiring expression of said selectable markergene for plant growth. Exemplary selectable marker genes and thecorresponding selective agents have been described in preceding sectionsof this description of the invention.

Transgenic plants can also be obtained by linking a gene of interest toa scoreable marker gene, introducing the linked transgenes into a plantcell by any one of the methods described above, and regenerating thetransgenic plants from transformed plant cells that test positive forexpression of the scoreable marker gene. Exemplary scoreable markergenes have been described in preceding sections of this description ofthe invention.

The regeneration, development and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art. See generally, Maliga et al., Methods in PlantMolecular Biology, Cold Spring Harbor Press (1995); Weissbach andWeissbach, In: Methods for Plant Molecular Biology, Academic Press, SanDiego, Calif. (1988). Plants of the present invention can be part of orgenerated from a breeding program, and may also be reproduced usingapomixis. Methods for the production of apomictic plants are known inthe art. See, e.g., U.S. Pat. No. 5,811,636.

It is not intended that the present invention be limited to theillustrated embodiments.

Crosses of Soybean Plants Containing Transgenes

In addition to direct transformation of a particular plant genotype witha construct prepared according to the current invention, transgenicplants may be made by crossing a plant having a selected DNA of theinvention to a second plant lacking the DNA. A selected DNA construct orconstructs that yield a high stearate/low linolenic acid/increased oleicacid phenotype can be introduced into a particular plant variety bycrossing, without the need for ever directly transforming a plant ofthat given variety. Therefore, the current invention not onlyencompasses a plant directly transformed or regenerated from cells whichhave been transformed in accordance with the current invention, but alsothe progeny of such plants. As used herein the term “progeny” denotesthe offspring of any generation of a parent plant prepared in accordancewith the instant invention, wherein the progeny comprises a selected DNAconstruct prepared in accordance with the invention. “Crossing” a plantto provide a plant line having one or more added transgenes or allelesrelative to a starting plant line, as disclosed herein, is defined asthe techniques that result in a particular sequence being introducedinto a plant line by crossing a starting line with a donor plant linethat comprises a transgene or allele of the invention. To achieve thisone could, for example, perform the following steps: (a) plant seeds ofthe first (starting line) and second (donor plant line that comprises adesired transgene or allele) parent plants; (b) grow the seeds of thefirst and second parent plants into plants that bear flowers; (c)pollinate a flower from the first parent plant with pollen from thesecond parent plant; and (d) harvest seeds produced on the parent plantbearing the fertilized flower.

Backcrossing is herein defined as the process including the steps of:(a) crossing a plant of a first genotype containing a desired gene, DNAsequence or element to a plant of a second genotype lacking said desiredgene, DNA sequence or element; (b) selecting one or more progeny plantcontaining the desired gene, DNA sequence or element; (c) crossing theprogeny plant to a plant of the second genotype; and (d) repeating steps(b) and (c) for the purpose of transferring a desired DNA sequence froma plant of a first genotype to a plant of a second genotype.

Introgression of a DNA element into a plant genotype is defined as theresult of the process of backcross conversion. A plant genotype intowhich a DNA sequence has been introgressed may be referred to as abackcross converted genotype, line, inbred, or hybrid. Similarly a plantgenotype lacking the desired DNA sequence may be referred to as anunconverted genotype, line, inbred, or hybrid.

Products of the Present Invention

The plants of the present invention may be used in whole or in part.Preferred plant parts include reproductive or storage parts. The term“plant parts” as used herein includes, without limitation, seed,endosperm, ovule, pollen, roots, tubers, stems, leaves, stalks, fruit,berries, nuts, bark, pods, seeds and flowers. In a particularlypreferred embodiment of the present invention, the plant part is a seed.

Any of the plants or parts thereof of the present invention may beprocessed to produce a feed, meal, protein, or oil preparation. In apreferred embodiment of the present invention can be a plant of thepresent invention having an oil with a fatty acid composition of thepresent invention. A particularly preferred plant part for this purposeis a seed. In a preferred embodiment the feed, meal, protein or oilpreparation is designed for livestock animals, fish or humans, or anycombination. Methods to produce feed, meal, protein and oil preparationsare known in the art. See, e.g., U.S. Pat. Nos. 4,957,748, 5,100,679,5,219,596, 5,936,069, 6,005,076, 6,146,669, and 6,156,227. In apreferred embodiment, the protein preparation is a high proteinpreparation. Such a high protein preparation preferably has a proteincontent of greater than 5% w/v, more preferably 10% w/v, and even morepreferably 15% w/v.

In a preferred oil preparation, the oil preparation is a high oilpreparation with an oil content derived from a plant or part thereof ofthe present invention of greater than 5% w/v, more preferably 10% w/v,and even more preferably 15% w/v. In a preferred embodiment the oilpreparation is a liquid and of a volume greater than 1, 5, 10 or 50liters. The present invention provides for oil produced from plants ofthe present invention or generated by a method of the present invention.Such oil may exhibit enhanced oxidative stability. Also, such oil may bea minor or major component of any resultant product.

Moreover, such oil may be blended with other oils. In a preferredembodiment, the oil produced from plants of the present invention orgenerated by a method of the present invention constitutes greater than0.5%, 1%, 5%, 10%, 25%, 50%, 75% or 90% by volume or weight of the oilcomponent of any product. In another embodiment, the oil preparation maybe blended and can constitute greater than 10%, 25%, 35%, 50% or 75% ofthe blend by volume. Oil produced from a plant of the present inventioncan be admixed with one or more organic solvents or petroleumdistillates.

Seeds of the plants may be placed in a container. As used herein, acontainer is any object capable of holding such seeds. A containerpreferably contains greater than about 500, 1,000, 5,000, or 25,000seeds where at least about 10%, 25%, 50%, 75% or 100% of the seeds arederived from a plant of the present invention. The present inventionalso provides a container of over about 10,000, more preferably about20,000, and even more preferably about 40,000 seeds where over about10%, more preferably about 25%, more preferably 50% and even morepreferably about 75% or 90% of the seeds are seeds derived from a plantof the present invention. The present invention also provides acontainer of over about 10 kg, more preferably about 25 kg, and evenmore preferably about 50 kg seeds where over about 10%, more preferablyabout 25%, more preferably about 50% and even more preferably about 75%or 90% of the seeds are seeds derived from a plant of the presentinvention.

Soybean seeds produced by the methods of the invention can comprisevarious oil compositions. An oil produced by soybean seeds produced bythe methods of the invention are referred to below as an “oil of thepresent invention”.

An oil of the present invention has a high stearate/low linolenicacid/increased oleic acid composition. In other embodiments, atransgenic oil of the present invention has increased stearate levelsand reduced linolenic acid levels. The percentages of fatty acidcontent, or fatty acid levels, used herein refer to percentages byweight.

In a first embodiment, a non-hydrogenated oil of the present inventionhas an oil composition that comprises greater than 35% oleic acid,greater than 10% stearate and less than 2% linolenic acid. In otherembodiments, the stearic acid content is greater than 17%, 25% and/or30% of total fatty acids by weight.

In a second embodiment, a soybean oil produced from a transgenic soybeanseed of the present invention has an oil composition that is greaterthan 10% stearate and less than 5% linolenic acid of total fatty acidsby weight.

In a third embodiment, a soybean oil produced from a transgenic soybeanseed of the present invention has an oil composition that is greaterthan 20% stearate and less than 1.7% linolenic acid of total fatty acidsby weight.

In a fourth embodiment, a soybean oil produced from a transgenic soybeanseed produced from a transgenic soybean seed I of the present inventionhas an oil composition that is greater than 30% stearate and less than2% linolenic acid of total fatty acids by weight.

In a fifth embodiment, a soybean oil produced from a transgenic soybeanseed of the present invention has an oil composition that is greaterthan 32% stearate and less than 5% linolenic acid of total fatty acidsby weight.

In a sixth embodiment, a soybean oil produced from a transgenic soybeanseed of the present invention has an oil composition that is greaterthan 10% stearate and between 39 and 57% oleic acid of total fatty acidsby weight.

In a seventh embodiment, a soybean oil produced from a transgenicsoybean seed of the present invention has an oil composition that isbetween 10 and 28% stearate, between 25 and 57% oleic acid and less than3% linolenic acid of total fatty acids by weight.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1 Transformation of Soybean A3525 with pMON97616, pMON97617,pMON97620, pMON97623 and pMON97624, and Resultant Phenotypes

Part I: Vector Descriptions pMON97616

Transgenic soybean plants were generated by an Agrobacterium-mediatedtransformation of soybean cells with the plasmid pMON97616 (FIG. 1).This binary plant transformation vector contains two planttransformation cassettes or T-DNAs. Each T-DNA is flanked by rightborder and left border sequences at the 5′ and 3′ ends of thetransformation cassette, respectively. The first T-DNA (SEQ ID NO: 3) isused for the expression of an inverted repeat designed to trigger theRNAi based suppression of endogenous FAD2 and FAD3 genes and also forthe expression of the FATA gene from Garcinia mangostana (SEQ ID NO: 2).The first T-DNA contains two expression cassettes and is organized asfollows: the first cassette is comprised of the nopaline RB sequence(basepairs (bp) 1-357), followed by a promoter from the Glycine max 7Salpha prime subunit of the beta-conglycinin gene (bp 419-620), which isupstream of the FATA gene from Garcinia mangostana (bp 632-1690), whichis upstream of the 3′ UTR (bp 1696-2008) of the Brassica rapa napingene. The second cassette on the T-DNA begins with the promoter (bp2058-2885) from the Glycine max7S alpha prime subunit ofbeta-conglycinin gene, followed by an inverted repeat (bp 2932-4407)containing 221 bp of the FAD2 intron and the 5′ and 3′ UTRs from twofamily members of the FAD3 gene family, which is upstream of the 3′ UTRof the Gossypium barbadense (bp 4451-4886) Sea island cotton H6 gene,which is upstream of the octopine LB sequence (bp 4946-5387). The 2^(nd)T-DNA contains the gene cassette conferring glyphosate resistance usedas the transformation selectable marker.

pMON97617

Transgenic soybean plants were generated by an Agrobacterium-mediatedtransformation of soybean cells with the plasmid pMON97617 (FIG. 2).This binary plant transformation vector contains two planttransformation cassettes or T-DNAs. Each T-DNA is flanked by rightborder and left border sequences at the 5′ and 3′ ends of thetransformation cassette, respectively. The first T-DNA (SEQ ID NO: 4) isused for the expression of an inverted repeat designed to trigger theRNAi based suppression of endogenous FAD3 genes and also for theexpression of the FATA gene from Garcinia mangostana. The first T-DNAcontains two expression cassettes and is organized as follows: the firstcassette is comprised of the nopaline RB sequence (bp 1-357), followedby a promoter from the Glycine max7S alpha prime subunit of thebeta-conglycinin gene (bp 419-620), which is upstream of the FATA genefrom Garcinia mangostana (bp 632-1690), which is upstream of the 3′ UTR(bp 1696-2008) of the Brassica rapa napin gene. The second cassette onthe T-DNA begins with the promoter (bp 2058-2898) from the Glycine max7Salpha prime subunit of beta-conglycinin gene, followed by an invertedrepeat (bp 2932-3965) containing the 5′ and 3′ UTRs from two familymembers of the FAD3 gene family, which is upstream of the 3′ UTR (bp4009-4444) of the Gossypium barbadense (Sea island cotton) H6 gene,which is upstream of the octopine LB sequence (bp 4504-4945). The 2^(nd)T-DNA contains the gene cassette conferring glyphosate resistance usedas the transformation selectable marker.

pMON97620

Transgenic soybean plants were generated by an Agrobacterium-mediatedtransformation of soybean cells with the plasmid pMON97620 (FIG. 3).This binary plant transformation vector contains two planttransformation cassettes or T-DNAs. Each T-DNA is flanked by rightborder and left border sequences at the 5′ and 3′ ends of thetransformation cassette, respectively. The first T-DNA (SEQ ID NO: 5) isused for the expression of an inverted repeat designed to trigger theRNAi based suppression of endogenous endogenous FAD2 and FAD3 and alsofor the expression of the FATA gene from Garcinia mangostana. The firstT-DNA contains two expression cassettes and is organized as follows: thefirst cassette is comprised of the nopaline RB sequence (bp 1-357),followed by a promoter (bp 419-620) from the Glycine max7S alpha primesubunit of the beta-conglycinin gene, which is upstream of the FATA genefrom Garcinia mangostana (bp 632-1690) which is upstream of the 3′ UTR(bp 1696-2008) of the Brassica rapa napin gene. The second cassette onthe T-DNA begins with the promoter (bp 2058-2898) from the Glycine max7Salpha prime subunit of beta-conglycinin gene, followed by an invertedrepeat (bp 2932-5171) containing 321 bp of the FAD2 intron, and the 5′and 3′ UTRs from three family members of the FAD3 gene family, which isupstream of the 3′ UTR (bp 5215-5650) of the Gossypium barbadense (Seaisland cotton) H6 gene, which is upstream of the octopine LB sequence(bp 5710-6151). The 2^(nd) T-DNA contains the gene cassette conferringglyphosate resistance used as the transformation selectable marker

pMON97623

Transgenic soybean plants were generated by an Agrobacterium-mediatedtransformation of soybean cells with the plasmid pMON97623 (FIG. 4).This binary plant transformation vector contains two planttransformation cassettes or T-DNAs. Each T-DNA is flanked by rightborder and left border sequences at the 5′ and 3′ ends of thetransformation cassette, respectively. The first T-DNA (SEQ ID NO: 6) isused for the expression of an inverted repeat designed to trigger theRNAi based suppression of endogenous FAD2 and FAD3 and also for theexpression of the FATA gene from Garcinia mangostana. The first T-DNAcontains two expression cassettes and is organized as follows: the firstcassette is comprised of the nopaline RB sequence (bp 1-357), followedby the promoter region (bp 403-1150) from the Brassica rapa napin gene,which is upstream of the FATA gene from Garcinia mangostana (bp1170-2228), which is upstream of the 3′ UTR (bp 2234-2546) of theBrassica rapa napin gene. The second cassette on the T-DNA begins withthe promoter (bp 2596-3436) from the Glycine max7S alpha prime subunitof beta-conglycinin gene, followed by an inverted repeat (bp 3470-5709)containing 321 bp of the FAD2 intron, and the 5′ and 3′ UTRs from threefamily members of the FAD3 gene family, which is upstream of the 3′ UTR(bp 5753-6188) of the Gossypium barbadense (Sea island cotton) H6 gene,which is upstream of the octopine LB sequence. The 2^(nd) T-DNA containsthe gene cassette conferring glyphosate resistance used as thetransformation selectable marker

pMON97624

Transgenic soybean plants were generated by an Agrobacterium-mediatedtransformation of soybean cells with the plasmid pMON97624 (FIG. 5).This binary plant transformation vector contains two planttransformation cassettes or T-DNAs. Each T-DNA is flanked by rightborder and left border sequences at the 5′ and 3′ ends of thetransformation cassette, respectively. The first T-DNA (SEQ ID NO: 7) isused for the expression of an inverted repeat designed to trigger theRNAi based suppression of endogenous FAD2 and FAD3 and also for theexpression of the FATA gene from Garcinia mangostana. The first T-DNAcontains two expression cassettes and is organized as follows: the firstcassette is comprised of the nopaline RB sequence (bp 1-357), followedby the promoter region (bp 421-1507) of the Lesquerella fendleri oleatedesaturase gene AH12, which is upstream of the FATA gene from Garciniamangostana (bp 1519-2577), which is upstream of the 3′ UTR (bp2583-2895) of the Brassica rapa napin gene. The second cassette on theT-DNA begins with the promoter (bp 2945-3785) from the Glycine max7Salpha prime subunit of beta-conglycinin gene, followed by an invertedrepeat (bp 3819-5494) containing 321 bp of the FAD2 intron, and the 5′and 3′ UTRs from three family members of the FAD3 gene family, which isupstream of the 3′ UTR (bp 5538-5937) of the Gossypium barbadense (Seaisland cotton) H6 gene, which is upstream of the octopine LB sequence(bp 6033-6474). The 2^(nd) T-DNA contains the gene cassette conferringglyphosate resistance used as the transformation selectable marker

Part II: Event Selection

Explants transformed with pMON97616, pMON97617, pMON97620, pMON97623 andpMON97624 were obtained via Agrobacterium tumefaciens-mediatedtransformation. Plants were regenerated from transformed tissue. 155 R0transformation events were carried forward after testing for 1-2 copiesof the H6 UTR fragment using Invader® (Third Wave Technologies, Inc.,Madison, Wis.). These events were self-pollinated to generate R1 seed.The fatty acid compositions of the R1 seeds were determined by FAME-GCanalysis. Pooled samples were ground to a fine powder and lipids wereextracted with heptane. The supernatant was transferred in a glass vialand the heptane was evaporated with a flow of dry nitrogen gas at 80° C.An aliquot of the extracted soybean oil (8 mg) was transesterified withsodium methoxide. Resultant fatty acid methyl esters (FAMEs) wereseparated by capillary gas chromatography and detected by flameionization detector. The column was a Supelcowax™ 10 with dimensions of15 m×0.25 mm×0.25 μm film thickness (Sigma-Aldrich, St. Louis, Mo.). Aninjection volume of 1 μl was used with a split ratio of 100:1. Peakswere identified based on their relative retention time compared to aFAME reference mixture. The resultant relative percent compositions ofthe major fatty acid components are reported (Table 1). Since the R1seed are segregating for the insertion, six individual seed wereanalyzed for each event to look at segregation and the single seed withthe highest stearate level was used as an early estimate of thehomozygous phenotype.

TABLE 1 Fatty Acid Composition of R1 Seeds Event Construct 16:0 18:018:1 18:2 18:3 GM_A433185 pMON97617 7.7 47.56 5.85 32.65 3.66 GM_A446735pMON97616 7.52 46.93 26.4 14.62 1.22 GM_A435769 pMON97620 7.43 46.9230.61 10.59 0.87 GM_A435767 pMON97620 5.66 45.46 38.66 6.53 0.73GM_A446764 pMON97616 6.23 43.24 6.89 33.78 7.28 GM_A435982 pMON976207.05 42.36 37.59 8.97 0.82 GM_A446714 pMON97616 6.44 42.15 23.98 23.251.78 GM_A435979 pMON97620 5.6 41.92 39.17 9.58 0.77 GM_A433338 pMON976177.26 38.97 9.01 40.72 1.55 GM_A446749 pMON97616 5.05 38.01 40.76 13.131.14 GM_A432204 pMON97624 6.3 38.01 9.59 36.24 7.59 GM_A446724 pMON976164.93 38 43.6 9.89 0.87 GM_A436393 pMON97623 5.78 37.5 44.49 8.59 0.68GM_A435525 pMON97620 6.63 35.82 8.77 37.77 9.26 GM_A436016 pMON976205.58 35.56 36.68 18.34 1.01 GM_A436608 pMON97623 4.98 34.91 47.39 10.130.8 GM_A446718 pMON97616 7.16 34.13 10.37 36.78 9.88 GM_A433508pMON97617 6.73 34.03 10.3 45.31 1.33 GM_A436385 pMON97623 5.59 33.4645.04 12.39 0.98 GM_A436620 pMON97623 5.03 32.64 47.41 11.59 0.88GM_A432534 pMON97624 4.99 32.49 50.02 8.91 0.85 GM_A432088 pMON976244.37 32.29 50.51 9.51 0.95 GM_A432084 pMON97624 4.64 32.26 50.71 9.480.78 GM_A446745 pMON97616 5.44 31.25 48.12 11.82 1.04 GM_A435561pMON97620 5.55 31.23 49.25 10.74 0.84 GM_A446716 pMON97616 5.44 31.1243.54 16.31 1.37 GM_A446708 pMON97616 6.61 31.04 11.49 40.45 8.15GM_A432210 pMON97624 5.45 30.75 48.69 11.48 1.15 GM_A432525 pMON976244.88 30.71 52.25 8.94 0.81 GM_A432527 pMON97624 4.23 30.22 55.18 7.090.72 GM_A433487 pMON97617 6.89 29.93 10.56 49.12 1.19 GM_A435775pMON97620 5.46 29.6 51.98 9.27 1.04 GM_A432344 pMON97624 4.83 29.4151.17 11.23 0.98 GM_A436192 pMON97623 6.59 29.36 16.94 37.47 7.44GM_A436184 pMON97623 5.04 29.34 55.98 6.41 0.68 GM_A435773 pMON976205.49 29.24 50.88 11.19 0.85 GM_A432080 pMON97624 4.81 28.65 54.97 9.070.85 GM_A432197 pMON97624 5.72 28.13 45.77 17.11 1.21 GM_A432337pMON97624 4.42 27.87 53.79 10.67 0.92 GM_A432347 pMON97624 4.57 27.8652.83 11.36 1 GM_A432512 pMON97624 5.04 27.8 55.3 8.67 0.79 GM_A432225pMON97624 5.29 27.49 51.47 12.12 1.2 GM_A433184 pMON97617 7.26 27.4111.66 50.42 1.23 GM_A432070 pMON97624 5.04 27.27 50.56 13.85 0.93GM_A436392 pMON97623 6.93 27.21 12.16 43.69 7.88 GM_A432339 pMON976245.23 26.73 52.2 12.4 1.02 GM_A435541 pMON97620 6.97 26.69 9.82 45.61 8.7GM_A432346 pMON97624 4.56 26.67 53.85 11.42 0.98 GM_A432537 pMON976245.09 26.44 51.82 13.14 1.24 GM_A432068 pMON97624 6.63 26.42 13.08 44.087.86 GM_A432513 pMON97624 7.16 25.49 11.34 46.08 7.82 GM_A436176pMON97623 5.33 24.95 56.31 10.24 0.88 GM_A433502 pMON97617 7.06 24.8612.47 52.14 1.59 GM_A433212 pMON97617 6.99 24.64 12.08 52.89 1.31GM_A436628 pMON97623 5.15 24.11 54.98 12.88 0.96 GM_A432336 pMON976244.85 24.07 57.37 10.53 0.92 GM_A436195 pMON97623 5.29 24 52.7 14.66 1.14GM_A432222 pMON97624 4.99 23.6 56.8 11.52 0.85 GM_A432340 pMON97624 8.0523.16 11.6 46.73 8.47 GM_A432361 pMON97624 5.01 22.75 57.97 10.74 1.16GM_A432354 pMON97624 4.99 22.63 61.85 7.19 0.89 GM_A432199 pMON97624 4.822.61 57.3 12.84 0.86 GM_A436605 pMON97623 5.32 22.45 60.63 8.63 0.84GM_A433201 pMON97617 7.88 22.27 15.21 51.78 0.99 GM_A432220 pMON976245.26 21.35 59.58 10.72 0.92 GM_A433335 pMON97617 7.67 21.04 14.61 53.531.3 GM_A446712 pMON97616 6.44 20.97 40.64 27.74 2.34 GM_A436618pMON97623 5.68 18.9 61.04 11.47 0.95 GM_A436178 pMON97623 5.51 18.1165.24 8.44 0.72 GM_A432213 pMON97624 8.62 17.54 13.5 49.55 8.98GM_A432076 pMON97624 5.19 17.28 62.03 12.65 0.97 GM_A432356 pMON976246.03 17.19 58.93 14.51 1.33 GM_A433499 pMON97617 9.74 12.68 15.44 59.691.37 GM_A432221 pMON97624 10.1 12.64 14.32 51.13 10.35 GM_A436382pMON97623 6.76 10.73 68.11 11.69 0.82 GM_A446721 pMON97616 6.89 8.8968.72 12.66 1.15 GM_A436014 pMON97620 7.71 8.7 71.92 8.86 0.92GM_A432518 pMON97624 7.15 8.6 68.2 13.32 1.11 GM_A436003 pMON97620 8.258.46 66.17 13.9 1.44 GM_A436173 pMON97623 8.53 8.21 57.36 22.87 1.47GM_A432200 pMON97624 8.38 8.18 52.67 28.35 1.51 GM_A446762 pMON976168.53 7.98 63.08 17.51 1.44 GM_A436193 pMON97623 7.91 7.97 69.87 11.541.04 GM_A432536 pMON97624 8.9 7.84 56.31 23.57 1.48 GM_A436612 pMON976237.23 7.7 68.72 13.66 1.07 GM_A433194 pMON97617 12.25 7.05 12.29 65.471.51 GM_A435537 pMON97620 7.79 7.04 71.49 11 1.01 GM_A433340 pMON9761713.03 6.96 11.33 65.95 1.84 GM_A436175 pMON97623 9.29 6.57 62.42 18.81.28 GM_A435989 pMON97620 12.88 6.29 17.54 56.77 5.33 GM_A436002pMON97620 9.12 6.24 64.68 16.59 1.57 GM_A433490 pMON97617 11.98 6.1916.82 56.01 8.24 GM_A446736 pMON97616 10.66 6.15 40.59 39.37 2.46GM_A432229 pMON97624 8.63 6.08 66.3 16.2 1.36 GM_A432529 pMON97624 8.735.86 70.36 12.3 1.1 GM_A432332 pMON97624 8.18 5.27 69.22 14.4 1.38GM_A432077 pMON97624 11.02 5.11 16.53 57.18 9.11 GM_A433183 pMON9761711.38 5.08 14.27 59.3 9.29 GM_A432227 pMON97624 10.4 5.05 61.82 20.061.14 GM_A433345 pMON97617 12.63 4.79 16.89 56.22 8.89 GM_A436590pMON97623 11.72 4.72 19.92 54.46 8.59 GM_A446726 pMON97616 9.73 4.6759.1 22.9 2.38 GM_A432223 pMON97624 11.4 4.65 13.78 57.47 12.03GM_A432528 pMON97624 11.23 4.63 18.3 56.29 8.54 GM_A433208 pMON9761712.4 4.6 18.21 61.62 2.48 GM_A436400 pMON97623 12.75 4.56 15.33 56.3410.02 GM_A433494 pMON97617 12.32 4.52 15.51 56.77 10.2 GM_A435560pMON97620 11.71 4.46 13.96 57.41 11.98 GM_A433196 pMON97617 11.87 4.4415.09 66.52 1.48 GM_A432089 pMON97624 11.24 4.39 15.4 59.57 8.86GM_A433199 pMON97617 11.15 4.37 9.53 70.52 2.45 GM_A436624 pMON976239.76 4.33 26.92 53.57 4.83 GM_A432351 pMON97624 9.71 4.3 65.24 18.051.26 GM_A446754 pMON97616 11.81 4.27 18.95 56.16 8.3 GM_A435536pMON97620 11.5 4.21 14.02 59.28 10.34 GM_A432163 pMON97624 13.41 4.1716.73 56.05 9.36 GM_A432514 pMON97624 9.54 4.04 70.12 13.27 1.23GM_A432522 pMON97624 9.66 3.99 70.75 13.21 1.08 GM_A432509 pMON9762411.18 3.98 47.31 34.15 2.11 GM_A432167 pMON97624 11.35 3.98 15.98 58.419.35 GM_A433205 pMON97617 11.76 3.96 17.82 63.02 2.81 GM_A433481pMON97617 11.77 3.95 18.39 59.98 5.52 GM_A436170 pMON97623 9.29 3.873.46 10.72 1.01 GM_A436610 pMON97623 9.7 3.8 68.81 15.67 1.32GM_A433206 pMON97617 11.05 3.75 22.89 54.22 7.14 GM_A433198 pMON9761713.21 3.75 15.74 64.8 1.45 GM_A446711 pMON97616 9.42 3.74 64.71 19.111.66 GM_A435785 pMON97620 12.69 3.7 22.62 55.94 4.05 GM_A435778pMON97620 12.25 3.69 40.31 39.97 2.88 GM_A433485 pMON97617 12.2 3.6417.94 61.69 3.52 GM_A446753 pMON97616 10.5 3.63 50.01 30.6 2.82GM_A433204 pMON97617 12.63 3.62 14.13 66.87 1.83 GM_A432169 pMON9762412.52 3.61 18.35 57.28 7.32 GM_A446737 pMON97616 10.45 3.6 28.8 51.364.72 GM_A436398 pMON97623 9.01 3.58 72.92 12.04 1.12 GM_A433339pMON97617 13.01 3.51 16.17 63.19 3.09 GM_A436404 pMON97623 9.25 3.572.84 11.38 1.25 GM_A436591 pMON97623 10.06 3.47 65.18 18.67 1.6GM_A446758 pMON97616 11.16 3.47 58.17 24.61 1.72 GM_A435519 pMON976209.52 3.3 72.42 11.48 1.32 GM_A436011 pMON97620 10.81 3.27 37.27 44.692.84 GM_A435787 pMON97620 10.68 3.24 54.52 28.24 2.15 GM_A436595pMON97623 9.49 3.16 73.61 11.41 1.01 GM_A436194 pMON97623 9.86 3.0571.84 12.61 1.22 GM_A436383 pMON97623 10.74 3.03 70.2 13.29 1.24GM_A436185 pMON97623 9.8 3.03 70.02 14.21 1.47 GM_A432166 pMON9762411.19 2.98 69.23 14.13 1.33 GM_A436378 pMON97623 9.24 2.89 74.79 10.381.18 GM_A432516 pMON97624 9.85 2.88 72.36 12.19 1.19 GM_A436606pMON97623 10.29 2.87 56.06 27.8 2.17 GM_A436015 pMON97620 10.01 2.8563.15 20.46 1.96 GM_A436384 pMON97623 9.69 2.77 66.75 17.71 1.34GM_A432350 pMON97624 8.8 2.75 75.7 10.05 1.21 GM_A433501 pMON97617 10.132.74 43.4 38.62 4.15 GM_A435999 pMON97620 10.11 2.72 67.33 15.88 1.91

Suppression of the native FAD2 and FAD3 genes combined with theoverexpression of the FATA gene from Garcinia mangostana gave seed oilwith elevated stearate content, elevated oleic acid (OA) content,decreased linoleic acid (LA) content and decreased alpha linolenic acid(ALA) content. Using >6% as a measure for elevated stearate (HS), <5% asmeasure for lowered ALA (LL) and a variable measure of elevated oleiccontent (MO) as it is dependent on the stearate level, we generatedTable 2 which lists the numbers of events that fall into the variouscombinations of the three targeted fatty acid alterations of highstearate/increased oleic acid/low ALA. Seventeen events did not have analtered fatty acid profile and were classified as nulls. We observedstearate ranging from 2.72% up to 47.56%, OA from 5.85% up to 75.70% andALA from 11.98% down to 0.68%. We have also observed the combination ofall three modifications as well as intermediate combinations in singlesoy seed.

TABLE 2 Phenotype groups Event Phenotype Number HS/LL 14 HS/MO/LL 72 HS8 LL 10 MO/LL 34 Null 17

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. A soybean oil composition comprising an oleicacid content of 35% to 57%, a stearic acid content of at least 35%, anda linolenic acid content of 0.68% to 1.14% by weight of fatty acids,wherein said oil composition is a soybean oil that is non-hydrogenated,that has not been refined, that has not been processed, and that has notbeen blended.
 2. The soybean oil composition of claim 1 having an oleicacid content of 39 to 57%.
 3. The soybean oil composition of claim 1,wherein said oil composition is a liquid and of a volume greater than 50liters.
 4. A soybean oil composition comprising an oleic acid content of35% to 57%, a stearic acid content of at least 35%, and a linolenic acidcontent of 0.68% to 1.14% by weight of fatty acids, wherein said oilcomposition is non-hydrogenated, has not been blended, and has beendegummed.
 5. The soybean oil composition of claim 4 having an oleic acidcontent of 39 to 57%.
 6. The soybean oil composition of claim 4, whereinsaid oil composition is a liquid and of a volume greater than 50 liters.7. The soybean oil composition of claim 1 having a stearic acid contentof 35% to 45.5%.
 8. The soybean oil composition of claim 1 having astearic acid content of 35% to 45.5% and an oleic acid content of 39% to57%.
 9. The soybean oil composition of claim 4 having a stearic acidcontent of 35% to 45.5%.
 10. The soybean oil composition of claim 4having a stearic acid content of 35% to 45.5% and an oleic acid contentof 39% to 57%.